Macrocyclic Transmembrane Anion Transporters via a One-Pot Condensation Reaction

Article abstract of DOI:10.1021/acs.orglett.0c01699

Synthetic chloride transporters are potential therapeutic agents for cystic fibrosis and cancer. Reported herein are macrocyclic transmembrane chloride transporters prepared by a one-pot condensation reaction. The most efficient macrocycle possesses a fine balance of hydrophobicity for membrane permeation and hydrophilicity for ion recognition. The macrocycle transports chloride ions by forming channels in the membrane. Hydrogen bonds and anion-πinteractions assist chloride transport.

Full text of DOI:10.1021/acs.orglett.0c01699

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Letter
Macrocyclic Transmembrane Anion Transporters via a One-Pot
Condensation Reaction
Parichita Saha and Nandita Madhavan*
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ABSTRACT: Synthetic chloride transporters are potential ther-
apeutic agents for cystic fibrosis and cancer. Reported herein are
macrocyclic transmembrane chloride transporters prepared by a one-
pot condensation reaction. The most efficient macrocycle possesses a
fine balance of hydrophobicity for membrane permeation and
hydrophilicity for ion recognition. The macrocycle transports
chloride ions by forming channels in the membrane. Hydrogen
bonds and anion−π interactions assist chloride transport.
atural ion channels carefully maintain the cellular
Nconcentrations of anions. Chloride ions play an
important role in biological systems.1 Faulty chloride channels
are linked to diseases such as cystic fibrosis,2−4 epilepsy,5,6 and
Bartter syndrome.1,7−9 It is challenging to reconstitute natural
chloride channels outside the cellular environment. Therefore,
synthetic chloride transporters can be potential therapeutics or
model systems to understand ion transport mechanisms.10−15
Specifically, chloride channels have shown promise for
treatment of cystic fibrosis and inducing apoptosis in cancer
cells.16−23
Compounds with an affinity for anions via hydrogen
bonding, anion−π interactions, or halogen bonding are
attractive scaffolds for anion transport.13,24−30 Cyclic scaffolds
such as calixarenes,31,32 calixpyrroles,14,33,34 and peptides35,36
are particularly interesting, as they possess a well-defined cavity
for ion recognition. We have previously developed a C3-
symmetric triamide macrocycle appended with cholesterol
units for chloride transport.37 The one-pot synthetic route
afforded macrocycles with identical R groups attached. Herein
we report a C2-symmetric chiral macrocycle scaffold that was
readily synthesized by a one-pot condensation reaction
between serine and pyridine derivatives. The macrocycle has
an interesting cavity containing hydrogen-bond-donating NH
groups and electron-deficient aromatic units for anion binding.
The synthetic design enables the incorporation of two distinct
R groups on the macrocycle periphery. The methodology was
used to synthesize macrocycles 1−3 (Figure 1) with increasing
numbers of long alkyl chains. The length of the alkyl chains
was chosen such that they would span half of the lipid bilayer
in their extended conformation. The hydrophobicity of the
macrocycles increased in the order of 1−3, with calculated log
Figure 1. Macrocycles with varying hydrophobicity. The length of the
alkyl chains is same for macrocycles 2 and 3. R1 and R2 are depicted in
different colors to differentiate their modes of attachment to the
macrocycle.
Received: May 19, 2020
https://dx.doi.org/10.1021/acs.orglett.0c01699
© XXXX American Chemical Society
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P values of 0.1, 11.7, and 22.1, respectively.38 Macrocycle 2
with an optimal balance of hydrophobicity for membrane
insertion and hydrophilicity for ion transport was the most
efficient. The macrocycle was anion-selective because of the
participation of amide NH groups in hydrogen bonding and
the aromatic units in anion−π interactions. The macrocycle
showed a preference for chloride ions.
Macrocycles 1−3 were synthesized by a one-pot con-
densation reaction between appropriately functionalized serine
esters and dipicolinic acid derivatives (Scheme 1). Macrocycle
Scheme 1. Synthesis of Macrocycles
1 was synthesized in 43% yield by coupling of commercially
available dipicolinic acid (4) and protected serine ester 5 in the
presence of (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluro-
nium hexafluorophosphate (HBTU) and N,N-diisopropyle-
thylamine (DIEA) (Scheme 1a). In view of the higher
nucleophilicity of the amine, the mechanism potentially
involves the formation of the amide linkage to the pyridine
unit followed by the ester linkage. In order to validate the
structure of macrocycle 1, it was also synthesized in a stepwise
fashion (Scheme S1). Pyridyl dicarbonyl dichloride was
coupled with 2 equiv of TBS-protected serine methyl ester.
The adduct was deprotected and coupled with pyridyl
dicarbonyl dichloride to give macrocycle 1. Macrocycle 2
was synthesized in 32% yield by coupling of diacid 4 with
functionalized serine ester 6.39 Macrocycle 3 was synthesized
in 16% yield by coupling of ester 6 with diacid 9, which was
obtained by the reaction of acyl chloride 8 with 4-
hydroxydipicolinic acid (7) (Scheme 1b).39
Vesicles made from egg yolk phosphatidyl choline (EYPC)
and cholesterol (9:1) containing the pH-sensitive dye 8-
hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS)
were used to study ion transport through macrocycles 1−3
(Figure 2a).40,41 The macrocycles were allowed to equilibrate
with the vesicles at pH 7.2, and NaOH was subsequently
added to increase the external pH by 0.6 units. No significant
increase in the concentration of deprotonated HPTS− was
observed for any of the macrocycles, ruling out pathways that
lead to an increase in the concentration of HPTS− (Figure 2a).
Figure 2. Studies to illustrate ion selectivity and compare the ion
transport activities of macrocycles. (a) HPTS assay to assess ion
transport activity. (b) Lucigenin assay for halide transport. (c)
Chloride-selective electrode to assess chloride transport. The
macrocycle concentration was 54.1 μM.
Varying the countercation of the base MOH (M = K+, Li+) did
not lead to any increase in the ion transport activity (Figure
S1). The studies indicated that the macrocycles did not
transport cations. UV−vis spectroscopy in the presence of
M2+-sensitive arsenazo dye (M2+ = Ca2+, Zn2+, Cu2+) also ruled
out transport of divalent cations by the macrocycles (Figures
S3 and S4).42−44 Assays carried out using the halide-sensitive
dye lucigenin showed that macrocycles 2 and 3 were chloride
transporters (Figure 2b).45 Macrocycle 1 without long alkyl
chains did not show ion transport activity. The dialkylated
macrocycle 2 was a better chloride transporter than
tetraalkylated macrocycle 3. This observation was corroborated
using a chloride-selective electrode as the probe (Figure 2c).
B
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The ion transport mechanism was determined using the
most efficient macrocycle, 2. It was intriguing that although the
macrocycle transported chloride ions, no internal pH increase
was observed in the HPTS assay (Figure 2a). In contrast to the
other assays in Figure 2, the HPTS assay did not have a
chloride gradient. Hence, HPTS assays with varying internal
and external buffer solutions were carried out to understand
the role of anions in the transport process (Figures 3a and S5).
counterion. (Figure 3c). The ion transport rates were
unaffected by the nature of the cation, further substantiating
the fact that cations were not involved in the transport process.
Dose−response studies were carried out using the HPTS
assay with a chloride gradient (Figure S6).37 The plots were
fitted to the Hill equation (eq S2) to obtain the EC50 value, i.e.,
the concentration of macrocycle required for half the
maximum activity (Figure 4a). The EC50 value for macrocycle
Figure 3. Assays to determine the mechanism of chloride transport
(symport/antiport). (a) HPTS assays carried out with variation of the
internal and external buffers to compare rates of Cl− transport (54.1
μM macrocycle 2). (b) Comparison of chloride and nitrate transport
using the NMDG assay (54.1 μM macrocycle 2). (c) Lucigenin assay
to compare the effects of cations on chloride transport (21.64 μM
macrocycle 2).
Figure 4. Mode of macrocycle−lipid interaction. (a) Hill analyses of
macrocycles. (b) U-tube experiment to distinguish between carrier
and channel mechanisms (1 mM macrocycle in the organic phase).
(c) Possible modes of pore formation by macrocycle 2.
The rate constants for ion transport were obtained by fitting
the curves to a first-order exponential decay equation (eq S1).
The maximum k values were observed when vesicles contained
2 (29.2 μM) was found to be 2.3 times lower than that for
macrocycle 3 (69.4 μM). The Hill coefficient of 1.6 indicated
that on an average a single ion was associated with one
macrocycle. The macrocycle could function as a carrier, form a
monomolecular pore or a stacked pore to accomplish this. The
classical U-tube experiment was used to determine whether the
macrocycle behaved as an ion carrier in the membrane (Figure
4b). The U-tube contained two aqueous layers separated by an
organic phase. The donating aqueous arm of the U-tube
contained NaCl, and the receiving arm contained water.
Macrocycle 2 was dissolved in the organic phase. The
concentration of chloride ions in the receiving arm did not
increase with time, suggesting that macrocycle 2 did not
function as an ion carrier and presumably formed a pore in the
membrane. The computed electrostatic potential surface of
macrocycle 1 (Figure S7) shows that the aromatic units being
electron-deficient substituents can participate in anion−π
interactions. The structures of macrocycles 1 and 2 bound
with chloride ions (Figure S8) show that the amide NH bonds
in the macrocycle can selectively bind with chloride ions via
hydrogen bonding, similar to 2,6-diamidopyridyl-derived anion
receptors.47−50 The chloride selectivity can also be explained
by the trend of Cl¯ > Br¯ > I¯ seen for anion−π interactions.51
Cl− inside and NO3 outside (a in Figure 3a). This suggests
−
that chloride efflux is the driving force for influx of hydroxide
ions, which increases the internal pH. The transport rate was
lower when chloride was outside the vesicles and nitrate was
inside (b in Figure 3a), suggesting that the macrocycle
preferred to transport chloride over nitrate ions. The rates of
ion transport were lowest in the HPTS assays without an anion
gradient, (c and d in Figure 3a). To compare the rates of Cl−
−
and NO3 transport, the HPTS assay was carried out using a
buffer containing N-methyl-D-glucamine (NMDG) chloride or
nitrate (Figure 3b).46 The pH gradient was introduced in the
system by the addition of NMDGOH. The only cations
present in the medium were the large membrane-impermeable
NMDG ions and protons. Gramicidin A was introduced into
the system to carry out H+ efflux. In such a scenario, the
macrocycle behaves as a uniporter carrying out Cl− or NO3
−
transport exclusively. The rate of Cl− transport was found to be
higher than that of NO3− transport. This observation is also in
line with rate constants observed for the HPTS assay (Figure
3a). To rule out the possibility of metal−halide cotransport,
the lucigenin assay was carried out by varying the metal
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Two types of pores are proposed on the basis of the Hill
coefficient (Figure 4c). A single macrocycle with alkyl chains
on opposite sides could form the pore. This model is suggested
because the length of macrocycle 2 with the alkyl chains
stretched out on opposite sides is ca. 40 Å, which is the size of
the lipid bilayer (Figure S8b). The flexible alkyl chains can also
be on the same side of the macrocycle, which allows them to
stack. The mode of stacking in Figure 4c is proposed on the
basis of computational studies with the macrocycle dimer
(Figure S9). Four macrocycles could stack to form the pore on
the basis of the length of the dimer (ca. 24 Å). The ion can
hop from one macrocycle to the other across the lipid bilayer.
This model explains the lower activity of macrocycle 3, where
the larger number of alkyl chains could hamper macrocycle
stacking.
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AUTHOR INFORMATION
■
Corresponding Author
Nandita Madhavan − Department of Chemistry, Indian
Institute of Technology Bombay, Mumbai 400 076, India;
orcid.org/0000-0003-1251-3527; Email: nanditam@
chem.iitb.ac.in
Author
Parichita Saha − Department of Chemistry, Indian Institute of
Technology Bombay, Mumbai 400 076, India
Complete contact information is available at:
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
P.S. thanks IIT Bombay and IIT Madras for fellowships. N.M.
thanks DST-SERB (EMR/2016/007672), India, for financial
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